The investigation of marine toxins by immunoassay, in particular ciguatoxins, was initiated from around 1977 along with the development of radioimmunoassay. Ciguatera poisons can be collected in a trace amounts from nature. For example, from 850 moray eels, namely from 4 tons of moray eels, only 0.35 mg of main poisonous constituent ciguatoxin is collected. And also, since it is difficult to produce ciguatoxin by cultivation, production of antibodies against ciguatoxins has been hampered. Hokama et al. of Hawaii University prepared a protein conjugate by coupling of ciguatoxin (1 μg) with human serum albumin by carbodiimido method and immunized on mice with said conjugate as an antigen, and elicited a monoclonal antibody (Toxicon Vol. 15, 1977, page 317) that bound to ciguatoxin. However, the antibody exhibited strong cross-reactivity with okadaic acid, and the difference of affinity is only about 5 times (Journal of Clinical Laboratory analysis Vol. 6, 1992, page 54). Furthermore, the antibody showed cross-reactivity with brevetoxins, maitotoxin or palytoxin (Journal of AOAC International Vol. 81, 1998, page 727), however, detailed deta has not been reported. A reagent or a kit (Cigua Check TM) for the detection of fishes polluted with ciguatoxins by an immunoassay has been developed utilizing the Hokama's antibody.
At the earlier stage, the inventors of the present invention synthesized ABC ring fragment, the left end of ciguatoxin, and prepared three kinds of monoclonal antibodies using protein conjugate obtained by utilizing the ABC ring fragment as a synthetic hapten, however, these monoclonal antibodies showed very weak affinity to ciguatoxin (Synthesis, 1999, page 1431). Other groups have also examined the immunization of protein conjugates of synthetic hapten (JKLM ring fragment), however, they have not succeed to prepare monoclonal antibody yet (Toxicon Vol. 38, 2000, page 669).
In the above circumstance, the inventors of the present invention have designed and synthesized a synthetic hapten derived from IJKLM ring fragment, which is a partial structure of right end of ciguatoxin CTX3C. The inventors also have established a hybridoma (deposited under the accession number FMRM PB-8293) by immunization of mice with a protein conjugate of the synthetic hapten and thereby have succeeded to produce a monoclonal antibody, which has high specificity to ciguatoxin CTX3C, using said hybridoma. And said monoclonal antibody is named as 3D11 [Deposit date at IPOD (International Patent Organism Depositary), AIST (National Institute of Advanced Industrial Science and Technology) was Mar. 5, 2002 and was deposited by request for transference according to Budapest convention on Feb. 13, 2003 under accession number FMRM PB-8293]. Dissociation constant of said monoclonal antibody 3D11 to ciguatoxin CTX3C is 122 nm. Further, the cross-reactivity of said antibody with structurally related polycyclic ether type marine toxins was investigated. Very weak cross-reactivity of the antibody 3D11 with red-tide toxin brevetoxins was detected, in which the affinity with brevetoxin was less than one 350th of the Kd value compared to that with CTX3C.
Further, as the synthetic hapten which is used in order to obtain monoclonal antibody of ciguatoxins, conjugate is synthesized by conjugating the derivative prepared by introducing carboxylic acid linker to C16 site of ABC ring fragment of ciguatoxin with BSA or KLH which are carrier proteins, emulsified the conjugate in RIBI adjuvant and injected the emulsified conjugate to five Balb/c mice for 4 times and immunized these mice, then spleen is picked out from these mice, cells of said spleen are fused with myeloma cells, P3X63-Ag8.653, and obtain said antibody and reaction specificity of the antibody to the synthetic hapten is estimated (Synthesis 1999, No. SI. 1431-1436 ISSN 0039-7881). That is, using ciguatoxins, in particular, partial structure of ciguatoxin CTX3C, study of synthetic hapten using a part of ring fragment of synthetic ciguatoxin used for the production of monoclonal antibody which is useful for the investigation of ciguatoxin CTX3C by immunological method is carried out. Concerning above mentioned study, the inventors of the present invention has continued designing a synthetic hapten through which can be obtained monoclonal antibody whose affinity to ciguatoxin is further improved. Further, since ciguatoxin is a large molecular whose molecule length is approximately 3 nano meter, ABCDE ring fragment, which is the partial structure of left-hand terminus of ciguatoxin, is designed as the hapten and investigated the method for approach for the synthesis. By the method to immunize a mouse with the protein conjugate of this synthetic hapten, above mentioned hybridoma 10C9 deposited under accession number FERM PB-8292 [Deposit date at the IPOD (International Patent Organism Depositary), AIST (National Institute of Advanced Industrial Science and Technology) was Mar. 5, 2002 and was deposited by request for transference according to Budapest convention on Feb. 13, 2003 under accession number FMRM PB-8292] is prepared, and succeeded to produce above mentioned one monoclonal antibody which has high specificity to ciguatoxins using said hybridoma.
The subject of the present invention is basically to provide a kit to detect ciguatoxins by sandwich method whose detective feature is more improved by using combination of two kinds of monoclonal antibodies. Further, for the purpose to accomplish said basic subject, the subject of the present invention is to provide two kinds of hybridomas which produce each monoclonal antibody to obtain said kit, two kinds of synthetic haptens useful to obtain each hybridoma and synthetic hapten-protein conjugates useful to obtain hybridoma which produce said monoclonal antibody prepared by combining the synthetic hapten to carrier protein.
As the first step, for the purpose to produce two kinds of monoclonal antibodies used for the sandwich method, two kinds of novel compounds used as synthetic haptens, one of them having IJKLM ring of composing ciguatoxin CTX3C and the other having ABCDE ring fragment of CTX3C are respectively synthesized.
Then, by immunizing with each protein-hapten product obtained by reacting each synthetic hapten with carrier protein, hybridoma which produce each monoclonal antibody reacting specifically to ciguatoxins CTX3C is obtained. And to one of said monoclonal antibody a labeling compound is bonded and by combining the labeled monoclonal antibody with other non labeled monoclonal antibody, sandwich immunoassay kit which can reduce false positive remarkably can be obtained and the technical concept of said sandwich immunoassay kit is established.